A positive energy balance that occurs when energy intake exceeds energy expenditure is an essential physiological condition for the development obesity. While much is known of the basic mechanisms controlling food intake, almost nothing is known of the identity of thermogenic mechanisms, apart from physical activity, that could be activated to burn off excess calories. We have forced mice, in which the mitochondrial uncoupling protein1 (UCP1) has been ablated, to activate alternative mechanisms of thermogenesis by gradually exposing them to the cold. Using several genetic models of UCP1 deficiency we have found that a novel gene, Slc25a25, has been consistently induced in skeletal muscle and inguinal fat under conditions of cold stress and diet-induced obesity (DIO). Since Slc25a25 encodes a putative ATP-Mg2+/Pi transporter, which is located on the inner mitochondrial membrane where it is thought to be involved in the regulation of ATP levels in the mitochondria, we have pursued experiments to test the hypothesis that Slc25a25 is involved in energy homeostasis. Mice with a global inactivation of Slc25a25 are resistant to DIO and have reduced physical endurance when tested on a treadmill. Based upon the fact that SLC25A25 has Ca2+-binding EF-hand domains and its ATP-Mg2+/Pi activity is regulated by Ca2+, we have designed experiments to test the hypothesis that SLC25A25 plays a crucial role in regulation of optimal energy levels for muscle excitation- contraction. In addition, a second gene, Gdm, which encodes the mitochondrial glycerol 3-phosphate dehydrogenase and has many of the same phenotypes and properties as SLC25A25, including its location in the inner mitochondrial membrane and possession of Ca2+-binding EF-hand domains, will be tested for its role in energy homeostasis. Three specific aims will test the hypothesis that SLC25A25 and GDM function as important components of energy homeostasis in skeletal muscle and heart by maintaining the level of ATP necessary to support Ca2+ cycling across the sarcoplasmic reticulum. In the first aim Slc25a25 will be inactivated selectively in heart and skeletal muscle using the Cre-LoxP system. DIO and physical endurance will be determined as well as fiber type analysis and transcriptome analysis of muscle to determine the effects of Slc25a25 inactivation on expression of genes involved in substrate oxidation and energy metabolism. The second aim will use ex vivo analysis of the perfused heart and skeletal muscle to determine the effects of inactivated Slc25a25 and Gdm on force characteristics of muscle under conditions of altered nutrition. The third aim will utilize mouse embryonic fibroblasts, prepared from mice with inactivated Slc25a25 and Gdm, to investigate the effects of mutant genes on Ca2+ imaging, respiration and ATP production in cells and isolated mitochondria using the Seahorse XF Extracellular Flux analyzer. These studies will establish whether SCL25A25 and GDM support Ca2+ cycling through maintenance of ATP levels and that their inactivation leads to metabolic inefficiency with effects on both muscle physical endurance and adiposity.